Passive Immunity to Feline Leukemia: Evaluation of Immunity from Dams Naturally Infected and Experimentally Vaccinated

INFECTION AND IMMUNITY, Apr. 1977, p. 54-59 Copyright C 1977 American Society for Microbiology Vol. 16, No. 1 Printed in U.S.A. Passive Immunity to Feline Leukemia: Evaluation of Immunity from Dams Naturally Infected and Experimentally Vaccinated EDWARD A. HOOVER,* JOSEPH P. SCHALLER, LAWRENCE E. MATHES, AND RICHARD G. OLSEN Department of Veterinary Pathobiology, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210 Received for publication 27 October 1976 Antibodies against feline leukemia virus (FeLV) and the feline oncornavirusassociated cell membrane antigen (FOCMA) were transferred from pregnant cats to their suckling kittens. All of these kittens were protected against infection and oncogenesis by virulent FeLV when challenged at 2 weeks of age. Suckling kittens acquired 25 to 100% of maternal virus-neutralizing and FOCMA titers by 3 days of age, and titers underwent linear decay to undetectable levels by 2 to 3 months of age. FOCMA antibody in dams and kittens was identified as immunoglobulin G (IgG) by use of goat anti-human IgG serum, which cross-reacts with feline IgG in the indirect membrane immunofluorescence test for FOCMA antibody. In an attempt to induce protective maternal antibody by vaccination, 10 pregnant cats were immunized by three to five weekly intramuscular injections with purified FeLV inactivated by ultraviolet irradiation. After the course of immunization, neither virus-neutralizing nor FOCMA antibody was detectable in the dams or in 19 kittens born to these cats. When these kittens were challenged with FeLV at 2 weeks of age, 18 of 19 developed persistent viremia and FeLV-related disease. Two approaches currently being evaluated for control of feline leukemia virus (FeLV) infection are (i) elimination of infected cats and (ii) immunoprophylaxis of susceptible cats. Because FeLV is transmitted horizontally among outbred cats (10, 14), both approaches are feasible. Hardy et al. (9) have shown that testing and removal of actively infected (viremic) cats is effective in reducing or eliminating FeLV infection and disease in test populations of cats. Although a limitation of this approach is the need for constant monitoring and control of the population to insure continual exclusion of FeLV-infected cats, the data have established conclusively that feline leukemia has epidemiological characteristics of a contagious disease. Vaccination of cats, therefore, is a logical alternative, particularly since humoral immunity appears to be important in resistance to feline oncornaviruses (1-5, 21). Active humoral immunity against the feline oncornavirus-associated cell membrane antigen (FOCMA) (2) develops in cats that resist natural or experimental infection with virulent FeLV (1-5, 21). It has been possible to induce active immunity to FOCMA by immunizing cats with killed FOCMA-containing tumor cells (15, 54 18, 19); however, comparable neutralizing antibody against FeLV or feline sarcoma virus (FeSV) has been difficult to induce in kittens with inactivated virus (19, 27). The latter is especially relevant, since pre-existing antibody to FOCMA does not necessarily protect cats from FeLV viremia (18, 19). Passively acquired FOCMA and virus-neutralizing (VN) antibody also protects against challenge with virulent FeSV, as shown by Essex et al. (2) for kittens suckling dams previously exposed horizontally to feline oncornaviruses and by Olsen et al. (19) for kittens suckling dams immunized with inactivated FeSV. One implication of these data is that since natural susceptibility to feline oncornaviruses is highest in the first 2 months of life (12, 25), vaccination of mother cats would confer passive immunity to suckling kittens during the period of maximal susceptibility. Subsequently, active immunization of weanling kittens could be initiated. The objectives of this study were (i) to evaluate an inactivated FeLV vaccine for its ability to induce humoral immunity in pregnant cats and thereby protect kittens born to these cats from challenge with virulent FeLV and (ii) to compare this immunity with that possessed by

VOL. 16, 1977 pregnant cats immunized by previous exposure to virulent FeLV. MATERIALS AND METHODS Cats. All cats were from a closed breeding colony of the Department of Veterinary Pathobiology, The Ohio State University. The cats are of hysterectomy-derived ancestry and are free of ectropic FeLV infection and FeLV-related diseases. Pregnant cats used for immunization were estimated by abdominal palpation to be between 28 and 35 days of gestation. Virus preparation and inactivation. The Kawakami-Theilen strain of FeLV (16) used for immunization of pregnant cats was harvested from a feline lymphoblast cell line (FL-74) (26). The FeLV-KT contains A-, B-, and C-subgroup specificities. Tenliter batches of FL-74 culture fluid were concentrated 10- to 20-fold using a Pellicon cassette system (Millipore Corp., Bedford, Mass.) with a 5-squarefoot (0.46-M2) cassette (10s nm/ia). Retentate fluids containing virus were then centrifuged at 56,700 x g over 50% ribonuclease-free sucrose (Schwarz/Mann, Div. of Becton, Dickinson & Co., Orangeburg, N.Y.) cushion prepared in TE buffer [0.1 ml of NaCl, 0.01 M tris(hydroxymethyl)aminomethane-hydrochloride (ph 7.2) and 0.001 M ethylenediaminetetraacetate]. Virus harvested from the cushion was then purified by two cycles of isopycnic banding in 0 to 50% sucrose (centrifugation for 18 h at 57,600 x g in a Beckman SW27 rotor; Beckman Instruments, Inc., Fullerton, Calif.). Virus bands were collected, dialyzed against TE buffer to remove sucrose, and stored in 2-ml portions at -70 C. Inactivation of purified virus was accomplished by ultraviolet (UV) irradiation at 150 ergs/mm2 per s for a total accumulated dose of 35,000 ergs/mm2 (27). The purified FeLV-KT contained between 5 x 1010 and 101" viral particles per ml, as determined by electron microscopic enumeration. The Rickard strain of FeLV (FeLV-R; subgroup A) (20) was used for oncogenic challenge of kittens. The inoculum consisted of a clarified 20% (wt/vol) homogenate of thymic lymphosarcoma tissue and contained 10W focus-forming units/ml as assayed in the 81 cell culture system originated by Fischinger et al. (6). The biological response of cats to the FeLV-R inoculum has been described (12). Immunization with UV-inactivated FeLV. Eight pregnant adult female cats were immunized with three to five weekly intramuscular injections of 1 ml of concentrated UV-inactivated FeLV-KT emulsified in an equal volume of Freund complete adjuvant. Injections were begun as soon as pregnancy was diagnosed (28 to 35 days) and continued until parturition occurred. Two nonpregnant adult female cats were given eight weekly immunizations with the same vaccine. Serum samples and blood smears were collected from each cat before each immunization. Immunization by natural exposure to virulent FeLV. Two female cars were exposed to virulent FeLV-R by contact with FeLV shed by suckling litters of kittens that had been inoculated with FeLV-R. Thus, the means of natural FeLV exposure PASSIVE IMMUNITY TO FeLV 55 duplicated that described by Essex et al. (2) for FeSV in adult female cats. Challenge of kittens. Kittens born to vaccinated or naturally immunized dams were challenged at 2 weeks of age by intraperitoneal injection of an established oncogenic dose of FeLV-R of tissue origin. The inoculum had been shown to produce an incidence of persistent viremia and FeLV-related disease (notably, thymic lymphosarcoma) in 85 to 100% of cats inoculated at less than 8 weeks of age (12). Serum samples and blood smears were collected from all kittens 2 days after birth and at biweekly intervals thereafter. All kittens were observed for evidence of FeLV-related disease for up to 6 months postchallenge. Serological tests. The presence of feline leukemia group-specific antigen (gsa) in circulating leukocytes was determined by the indirect immunofluorescence test described by Hardy et al. (8). The FeLV antiserum was of goat, rather than rabbit, origin (18). FOCMA antibody was assayed by the indirect membrane immunofluorescence (IMI) test described by Essex et al. (2). The immunoglobulin class of FOCMA antibody was determined by a tertiary (IMI) test in which either goat anti-human immunoglobulin G (IgG) or goat anti-human IgM serum (Hyland, Div. of Travenol Laboratories, Inc., Costa Mesa, Calif.) was substituted for the rabbit antifeline globulin serum (Sylvana, Millburn, N.J.) usually used as the secondary reagent in the IMI FOCMA test (2). The cross-reactivity and specificity of the anti-human IgG and IgM reagent with putative feline IgG and IgM were determined by immunoelectrophoresis against whole feline serum. A single line of precipitation formed in the region and shape corresponding to the migration of feline IgG and IgM (23) (Fig. 1). The tertiary reagent was fluorescein isothiocyanate-conjugated rabbit antigoat immunoglobulin (Hyland Laboratories). VN antibody against FeLV subgroup A was determined as described by Schaller and Olsen (22). RESULTS Adult female cats vaccinated with UV-inactivated FeLV. Of 10 pregnant cats vaccinated from three to five times over a period of 5 to 6 weeks, 9 failed to develop demonstrable VN antibody titers (i.e., < 1:2). One cat developed a titer of 1:16 after four immunizations but died of suppurative pyelonephritis prior to parturition. The VN titers of two nonpregnant cats also remained at <1:2 after eight weekly immunizations with UV-inactivated FeLV-KT (Table 1). Adult female cats exposed to virulent FeLV. Two cats that had suckled litters of FeLV-inoculated kittens developed VN titers of 128 and 64 (geometric mean = 90) and FOCMA antibody titers of 32 and 16 (geometric mean = 21), respectively (Table 2). Neither cat became FeLV gsa positive.

56 HOOVER ET AL. INFECT. IMMUN. Downloaded from http://iai.asm.org/ FIG. 1. Immunoelectrophoresis patterns with goat anti-human IgM (top trough) of anti-human IgG (bottom trough) versus whole feline serum. A single line ofprecipitation overlies the expected position of the arcs for feline IgM and IgG, respectively. Cathode at left. Kittens born to cats immunized with UVinactivated FeLV. Eighteen of 19 kittens born to six dams immunized with UV-inactivated FeLV became persistently FeLV gsa positive (viremic) after challenge with virulent FeLV. All of the 18 FeLV gsa-positive kittens developed FeLV-related disease in 6 to 35 weeks (Table 1). Twelve of the 18 cats developed thymic lymphosarcoma, and 6 died either of early undetermined causes (no necropsy possible) or of bacterial sepsis. The one cat to remain FeLV gsa negative after challenge developed persistent FOCMA and VN antibody titers and remained free ofdisease after 40 weeks ofobservation. Kittens born to cats immunized by virulent FeLV. By 3 days of age, all kittens had acquired VN and FOCMA antibody titers ranging from 25 to 100% of maternal titers. In all kittens, VN and FOCMA antibody titers decayed to <1:2 by 2 months of age (Table 2, Fig. 2). A secondary rise in both titers occurred between 4 and 5 months of age in all four kittens of one litter but in neither of two kittens in the second litter. At the time of FeLV challenge at 2 weeks of age, kittens had a geometric mean VN titer of 13 (range, 8 to 16), and the mean FOCMA titer was 8 (range, 4 to 16) (Table 2, Fig. 2). After FeLV inoculation, none of the six kittens became FeLV gsa positive and none developed disease after 7 months of observation (Table 2). Immunoglobulin class of FOCMA antibody in dams and kittens. Table 3 illustrates the IgG and IgM profiles of kittens with passively and actively acquired FOCMA antibody. In both the dams' and kittens' sera, the major FOCMA on April 24, 2019 by guest

VOL. 16, 1977 PASSIVE IMMUNITY TO FeLV 57 antibody was IgG. In most cases, IMI FOCMA IMI FOCMA test) or goat anti-human IgG was titers were within one twofold dilution of each used as the secondary reagent. In two of the other, whether rabbit antiserum against feline four kittens in which secondary FOCMA titers globulin (the usual secondary reagent in the developed, both initial (passive) and secondary TABLE 1. Response ofpregnant cats to immunization with UV-inactivated FeLV and susceptibility of their kittens to challenge with virulent FeLV Immunized pregnant cats Kittens born to immunized pregnant cats Serological data postim- Serological data pre- Serological data post-felv challenge munization (time of par- FeLV challengea (3 days (6 to 30 weeks of age) Ani- No. of turition) Ani- to 2 weeks of age) mal immuni- mal no. zations VN FOCMA FeLV no. VN FOCMA FeLV VN FOCMA FeLV FeLV-reanti- antibody gsa anti- antibody gsa anti- antibody gsa lated dis- body body body ease 83R 5 <2 2-83R-1 <2 <2 - <2 <2 + + -2 <2 <2 - NTb <2 + + -3 NT <2 - NT <2 + + -4 NT <2 - NT <2 + + 88R 5 <2 <2-88R-1 <2 <2 - <2 <2 + + -2 <2 <2 - NT <2 + + -3 NT <2 - NT <2 + + -4 NT <2 - NT <2 + + 152R 3 <2 <2-152R-2 <2 <2 - <2 <2 + + -4 NT <2 - NT <2 + + -5 NT <2 - NT <2 + + -6 NT <2 - NT <2 + + 895 5 <2 <2-895-2 <2 <2 - <2 <2 + + 921 5 <2 <2-921-1 <2 <2 - <2 <2 + + 964 5 <2 2-964-1 <2 <2 - <2 <2 + + -2 <2 <2 - NT <2 + + -3 <2 <2 - NT <2 + + -4 <2 <2 - <8 <8 822c 4 <2 <2 - None 890d 4 16 4 - None 81Be 8 <2 <2 - None 59B 1 8 <2 <2 - None a Challenge by intraperitoneal injection of a known oncogenic dose of FeLV-R at 2 weeks of age. b NT, Not tested. c Kittens stillborn. d Dam died of suppurative pyelonephritis before parturition. e Nonpregnant cat. TABLE 2. Passive transfer of VN and FOCMA antibody from mother cats immunized by exposure to virulent FeLV and susceptibility of their kittens to FeLV challenge Geometric mean titer FeLV gsa sta- FeLV-related Animal group (no.) Time after parturition tus disease VN FOCMAtudias Dams (2) 0 90 22 nege neg 2 weeksa NTb NT neg neg 1 mo 90 22 neg neg 3 mo 90 32 neg neg Kittens (6) 0 27.9 13.9 neg neg 2 weeksa 13.0 8.0 neg neg 1 mo 4.6 3.2 neg neg 3 mo 0.0 1.2 neg neg 6 mo 2.5 7.0 neg neg 7 mo 6.5 3.5 neg neg a FeLV challenge of kittens at 2 weeks of age. b NT, Not tested. e neg, Negative.

58 HOOVER ET AL. VN- DAMS INFECT. IMMUN. z 40 a 30 u FOCMA- DAMS w 2 20 0 w ' - 10 5. - IVNKITTENS "i<ocma- KITTENS.-...*' Pz,..FOM T T 2 3 4 DAY OF Fe LV AGE (MONTHS) 5 6 7 BIRTH CHALLENGE FIG. 2. Passive transfer of VN and FOCMA antibody from mother cats immunized by exposure to virulent FeLV to their suckling kittens. TABLE 3. Immunoglobulin class of FOCMA antibody in serial serum samples from a kitten (956-1) born to a dam with FOCMA antibody acquired by prior exposure to virulent FeLV Secondary reagent used to determine IMI Time after FOCMA titer birth (mo) Anti-feline Anti-IgG Anti-IgM globulina" niig At-g 0 32 NTb NT 1 4 8 2 2 2 2 16 3 2 4 2 5 16 32 2 7 32 32 2 a Reciprocal of highest positive dilution. " NT, Not tested; serum at this interval exhausted. titers were composed of IgG with trace amounts of IgM. In the remaining two kittens, putative IgM IMI FOCMA antibody was detected in the absence of IgG activity at 2 months of age (Table 3) when little or no FOCMA titer was demonstrable using the usual anti-feline immunoglobulin antiserum (which is directed almost exclusively toward feline IgG). DISCUSSION This report verifies that maternal transfer of protective levels of VN and FOCMA antibody occurs. The presence or absence of humoral antibody in dams correlated directly with resistance or susceptibility of their kittens to FeLV. One hundred percent of kittens with VN titers of at least 8 were protected against a known oncogenic FeLV challenge that proved fatal in 95% of kittens from dams without detectable VN titers. Our data concerning passive transfer of FOCMA antibody, resistance to oncogenic challenge, and the IgG nature of FOCMA antibody required by suckling kittens are similar to those obtained by Essex et al. (2) with FeSV. In addition, we found that decay of VN and FOCMA antibody in suckling kittens was virtually linear over a 2-month period, as expected for passively acquired antibody. Nomographs for maternal antibody to feline panleukopenia virus in kittens are similar, although feline panleukopenia virus titers are usually higher initially and thereby persist longer (24). Our data also document that neutralizing antibody to FeLV is difficult to induce with inactivated virus. Little or no VN antibody was demonstrable after as many as eight weekly injections of concentrated UV-inactivated FeLV-KT. These observations agree with those of Olsen et al. (19) and Yohn et al. (27) concerning inactivated FeSV vaccines and with those of Hardy et al. (11) concerning FeLV preparations inactivated with formalin. Some possible explanations for the cats' minimal immune response to inactivated FeLV include the following. (i) Amplification of antigenic mass or the direct exposure of lymphoreticular organs that are associated with FeLV replication in vivo are critical to development of an effective immune response; (ii) immunogenicity of the virus is compromised during purification or inactivation; and (iii) FeLV glycoproteins must be presented in association with a cell membrane complex for appropriate recognition and trig-

VOL. 16, 1977 PASSIVE IMMUNITY TO FeLV 59 gering of the VN antibody response. The inefficacy of inactivated FeLV as an immunogen is in contrast to the response of mice to inactivated Gross (13), Friend (7), and Rauscher (17) murine leukemia virus. Furthermore, mice suckling mothers vaccinated with formalized Gross or Friend murine leukemia virus were protected from homologous oncogenic challenge by passive maternal antibody (13, 17). In conclusion, the presence of maternal humoral immunity to FeLV is transferred to suckling kittens and is effective in protecting these kittens from FeLV infection and disease. A major limitation to immunization procedures involving inactivated FeLV was the difficulty in obtaining an immune response to nonreplicating virus. Other protocols aimed at improving the immune response of cats to killed FeLV are currently being evaluated. ACKNOWLEDGMENTS This study was supported by Public Health Service Contract NO1-CP-53571 within the Special Virus Cancer Program of the National Cancer Institute. We thank Steven Torvik, Dagmar Imel, Lilly Romvary, Les Takacs, Kenneth Milliser, and Steven Hunziker for their valuable contributions to completion of this work. LITERATURE CITED 1. Essex, M. 1975. Horizontally and vertically transmitted oncornaviruses of cats. Adv. Cancer Res. 21:175-248. 2. Essex, M., G. Klein, S. P. Snyder, and J. B. Harrold. 1971. Antibody to feline oncornavirus-associated cell membrane antigen in neonatal cats. Int. J. Cancer 8:384-390. 3. Essex, M., A. Sliski, W. D. Hardy, Jr., and S. M. Cotter. 1976. Immune response to leukemia virus and tumor-associated antigens in cats. Cancer Res. 36:640-645. 4. Essex, M., and S. P. Snyder. 1973. Feline oncornavirusassociated cell membrane antigen. I. Serologic studies with kittens exposed to cell-free materials from various feline fibrosarcoma. J. Natl. Cancer Inst. 51:1007-1012. 5. Essex, M., S. P. Snyder, and G. Klein. 1973. Relationship between humoral antibodies and the failure to develop progressive tumors in cats injected with feline sarcoma virus, p. 771-777. In R. M. Dutcher and L. Chieco-Bianchi (ed.), Unifying concepts of leukemia. S. Karger AG, Basel. 6. Fischinger, P. J., C. S. Blevins, and S. Nomura. 1974. Simple, quantitative assay for both xenotropic murine leukemia and ecotropic feline leukemia viruses. J. Virol. 14:177-179. 7. Friend, C. 1959. Immunological relationships of a filterable agent causing a leukemia in adult mice. 1. The neutralization of infectivity by specific antiserum. J. Exp. Med. 109:217-228. 8. Hardy, W. D., Jr., Y. Hirshaut, and P. Hess. 1973. Detection of the feline leukemia virus and other mammalian oncornaviruses by immunofluorescence, p. 778-799. In R. M. Dutcher and L. Chieco-Bianchi (ed.), Unifying concepts of leukemia. S. Karger AG, Basel. 9. Hardy, W. D., Jr., A. J. McClelland, E. E. Zuckerman, P. W. Hess, M. Essex, S. M. Cotter, E. G. MacEwen, and A. A. Hays. 1976. Prevention of contagious spread of the feline leukemia virus between pet cats, p. 180-183. In J. Clemmesen and D. S. Yohn (ed.), Comparative leukemia research 1975. Bibl. Haemat. no. 43, S. Karger AG, Basel. 10. Hardy, W. D., Jr., L. J. Old, P. W. Hess, M. Essex, and S. Cotter. 1973. Horizontal transmission of feline leukemia virus. Nature (London) 244:266-269. 11. Hardy, W. D., Jr., E. E. Zuckerman, A. J. McClelland, P. W. Hess, E. G. MacEwen, M. Essex, and S. M. Cotter. 1975. Feline leukemia virus control and vaccination, p. 152. In Proceedings of the VIIth International Symposium on Comparative Leukemia Research, Copenhagen, Denmark. 12. Hoover, E. A., R. G. Olsen, W. D. Hardy, Jr., J. P. Schaller, and L. E. Mathes. 1976. Feline leukemia virus infection: age-related variation in response of cats to experimental infection. J. Natl. Cancer Inst. 57:365-369. 13. Ioachim, H. L., M. L. Gimovsky, and S. E. Keller. 1973. Maternal vaccination with formalin-inactivated gross lymphoma virus in rats and transfer of immunity to offspring. Proc. Soc. Exp. Biol. Med. 144:376-379. 14. Jarrett, W., 0. Jarrett, L. Mackey, H. Laird, W. D. Hardy, Jr., and M. Essex. 1973. Horizontal transmission of leukemia virus and leukemia in the cat. J. Natl. Cancer Inst. 51:833-841. 15. Jarrett, W., 0. Jarrett, L. Mackey, H. Laird, C. Hood, and D. Hay. 1975. Vaccination against feline leukemia virus using a cell membrane system. Int. J. Cancer 16:134-141. 16. Kawakami, T. G., G. H. Theilen, D. L. Dungworth, R. J. Munn, and S. G. Beall. 1967. "C"-Type viral particles in plasma of cats with feline leukemia. Science 158:1049-1050. 17. Mirand, E. A., J. T. Grace, Jr., and R. F. Buffett. 1966. Passive and active immunity to Friend virus disease. Nature (London) 209:696-698. 18. Olsen, R. G., E. A. Hoover, L. E. Mathes, L. Heding, and J. P. Schaller. 1976. Immunization against feline oncornavirus disease using a killed tumor cell vaccine. Cancer Res. 36:3642-3646. 19. Olsen, R. G., J. P. Schaller, E. A. Hoover, and D. S. Yohn. 1976. Experimental oncornaviruses vaccines in the cat, p. 515-517. In J. Clemmesen and D. S. Yohn (ed.), Comparative leukemia research 1975. Bibl. Haemat. no. 43, S. Karger AG, Basel. 20. Sarma, P. S., and T. Log. 1973. Subgroup classification of feline leukemia and sarcoma viruses by viral interference and neutralization tests. Virology 54:160-169. 21. Schaller, J. P., M. Essex, D. S. Yohn, and R. G. Olsen. 1975. Feline oncornavirus-associated cell membrane antigen. V. Humoral immune response to virus and cell membrane antigens in cats injected with Gardner-Arnstein feline sarcoma virus. J. Natl. Cancer Inst. 55:1373-1378. 22. Schaller, J. P., and R. G. Olsen. 1975. Determination of subgroup-specific feline oncornavirus-neutralizing antibody. Infect. Immun. 12:1405-1410. 23. Schultz, R. D., F. W. Scott, J. R. Duncan, and J. H. Gillespie. 1974. Feline immunoglobulins. Infect. Immun. 9:391-393. 24. Scott, F. W., C. K. Csiza, and J. H. Gillespie. 1970. Maternally derived immunity to feline panleukopenia. J. Am. Vet. Med. Assoc. 156:439-453. 25. Snyder, S. P., and D. L. Dungworth. 1973. Pathogenesis of feline viral fibrosarcomas: dose and age effects. J. Natl. Cancer Inst. 51:793-798. 26. Theilen, G. H., T. G. Kawakami, J. D. Rush, and R. J. Munn. 1969. Replication of cat leukemia virus in cell suspension cultures. Nature (London) 222:589-590. 27. Yohn, D. S., R. G. Olsen, J. P. Schaller, E. A. Hoover, L. E. Mathes, L. Heding, and G. W. Davis. 1976. Experimental oncornavirus vaccines in the cat. Cancer Res. 36:646-651.